Cholera remains a major health problem in large parts of the world. This is also true for ETEC, which is the main cause of diarrheal disease in developing countries as well as in travelers to these countries. In many developing countries effective water and sanitary measures for control of cholera and other enteric infections are currently impossible, and in this context, vaccines have an important role to play. In order to do so however, they need to be effective, readily accessible and above all cheap. There is also a medical need and a very substantial commercial market for use of cholera and especially ETEC vaccines in travelers.
One approach has been the development of oral killed whole cell vaccines. The oral vaccine sold under the trademark, DUKORAL™ is an oral, monovalent inactivated vaccine containing killed whole cells of V. cholerae O1 plus additional recombinant cholera toxin B subunit with demonstrated up to 90% efficacy against cholera and also a significant efficacy against Enterotoxigenic Escherichia coli (ETEC)-induced diarrhea. It comprises 3 different V. cholerae strains in four different formulations (two heat-killed and two formalin-killed) and in addition recombinantly produced cholera toxin B subunit (rCTB). The rCTB component contributes significantly to the efficacy against cholera and is solely responsible for the observed protection against ETEC diarrhea due to its ability to induce cross-neutralizing antibodies against the cholera toxin (CT)-like E. coli heat-labile toxin (LT). However, rCTB is acid-labile and thus the vaccine (which needs to be given in two doses) must be administered with a bicarbonate buffer.
Despite DUKORAL™, which is an oral, monovalent inactivated vaccine containing killed whole cells of V. cholerae O1 plus additional recombinant cholera toxin B subunit, being the only internationally licensed OCV, copies of this vaccine with or without the CTB component are currently being marketed in developing countries—Vietnam, India and China. The OCV made in Vietnam and India (which lacks the CTB component) contains the same 4 bacterial components as in the oral vaccine sold under the trademark, DUKORAL™, which is an oral, monovalent inactivated vaccine containing killed whole cells of V. cholerae O1 plus additional recombinant cholera toxin B subunit, plus a fifth formalin-killed V. cholerae strain of serogroup O139.
Protective immunity against cholera elicited by OCVs is mainly if not exclusively based on mucosal production of antibodies against cell wall lipopolysaccharide O1 (O1 LPS) and for the CTB-containing the oral vaccine sold under the trademark, DUKORAL™, which is an oral, monovalent inactivated vaccine containing killed whole cells of V. cholerae O1 plus additional recombinant cholera toxin B subunit, also antitoxin antibodies in the intestine.
From the above it is evident that the present state of the art for production of cholera/ETEC vaccine is far from simple, and although already effective, a real contribution to making a cholera vaccine more accessible would be to rationalize the composition of the formulation at several levels.
The necessity to include several different Vibrio cholerae strains in killed whole cell vaccines arises from the need to represent several different antigenic variants of Vibrio cholerae in the vaccine. All protective strains in the currently used vaccines are of the O1 serogroup which until 1993 was the only one of more than 200 identified serogroups known to cause epidemic cholera and is still the dominant serogroup. However, the O1 serogroup has two variants called the Ogawa and Inaba serotypes that differ in the methylation of the terminal sugar of the O-antigen of the surface lipopolysaccharide (LPS). Serotype switching is known to occur in which the Ogawa serotype organism can give rise to Inaba organisms. The reverse switch however is rare.
Although immunization with especially Inaba but also Ogawa serotype can give rise to antibodies cross-reacting with the other serotypes it also gives rise to serotype specific antibodies that contribute significantly to protection. Thus, an effective vaccine should induce not only cross reactive but also serotype-specific antibodies against both Inaba and Ogawa serotype variants.
The serotype switch is known to be related to a mutation in a single gene (wbeT). Any mutation that inactivates this gene results in a switch from the Ogawa to the Inaba serotype. Mutations that can reverse such an event are predictably much more uncommon although a switch from the Inaba to the Ogawa serotype can easily be achieved by provision of the relevant gene in trans. The gene involved (wbeT, also denoted rfbT) encodes a methyl transferase that methylates the terminal perosamine residue in the O-antigen polysaccharide repeating unit. Mutations in this gene that lead to the Inaba serotype are almost invariably insertions, deletions or base changes that introduce a nonsense codon.
A third O1 variant known as Hikojima has also been documented to occur in the wild. Hikojima is characterized by that it expresses both the Ogawa and Inaba determinants on its surface and agglutinates with antisera specific for both types. The Hikojima phenotype is extremely rare and is considered in the literature to be an unstable transitional form.
With this in mind the inventors have set out to engineer a single vaccine strain of V. cholerae that would effectively replace the three currently used strains.
Thus, it is an object of the invention to provide an efficient vaccine against cholera and/or ETEC diarrhea, with simplified formulation and with lower productions costs and that also ideally produces protective immunity after single administration.